Bottom Line:
The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training.By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion.Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.

ABSTRACTSpatial water maze (WM) overtraining induces hippocampal mossy fiber (MF) expansion, and it has been suggested that spatial pattern separation depends on the MF pathway. We hypothesized that WM experience inducing MF expansion in rats would improve spatial pattern separation in the hippocampal network. We first tested this by using the the delayed non-matching to place task (DNMP), in animals that had been previously trained on the water maze (WM) and found that these animals, as well as animals treated as swim controls (SC), performed better than home cage control animals the DNMP task. The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training. Moreover, these behavioral treatments also enhance network reliability and improve partial pattern separation in CA1 and pattern completion in CA3. By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion. Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.

pone.0132676.g006: Synaptophysin/Map2 segmentation and Map2 staining area analysis.(A) A representative synaptophysin (Red) / Map2 (Green) stained image is shown with the drawings that defined the different hippocampal dendritic segments regions of interest (hippocampal segments ROIs). These hippocampal segments ROIs included the CA3 stratum oriens, divided into 3 regions based on their proximity to the dentate gyrus (DG): stratum oriens distal (SOd), stratum oriens medial (SOm), and stratum oriens proximal (SOp); the CA3 stratum lucidum was also divided into 3 regions, the stratum lucidum distal (SLd), stratum lucidum medial (SLm), and stratum lucidum proximal (SLp). The CA3 stratum radiatum was divided into 6 regions, depending on their proximity to the DG and to the pyramidal cell soma: stratum radiatum distal medial (SRdm), stratum radiatum medial medial (SRmm), stratum radiatum proximal medial (SRpm), stratum radiatum distal distal (SRdd), stratum radiatum medial distal (SRmd), and stratum radiatum proximal distal (SRpd). Finally, the last 2 ROIs correspond to the CA1 stratum oriens (CA1 SO) and the CA1 stratum radiatum (CA1 SR). In the bar graph (B) the Map2-stained area expressed in pixels is shown for each hippocampal segment ROI. It is important to emphasise that No significant differences were found among groups (IC, SC, and WM) in the Map2-stained area used for the synaptophysin analysis.

Mentions:
Analysis of the immunofluorescence for synaptophysin/Map2 staining was performed using ImageJ software (Freeware NIH USA). All images were pre-processed with the median filter for noise reduction. Then, each image was segmented into 14 different regions of interest (ROIs). The ROIs were drawn to segment different portions of the CA3 and CA1 dendritic regions (Fig 6A). In CA3, we distinguished between the stratum oriens and stratum lucidum, and between proximal and distal portions of the stratum radiatum based on their proximity to the CA3 pyramidal cell soma. The CA3 region was also divided into 3 regions (distal, medial, and proximal) based on their proximity to the DG granular layer (12 ROIs total in CA3). Finally, we divided the CA1 region between the stratum oriens (CA1 SO) and stratum radiatum (CA1 SR). It is possible that the 2 ROIs selected in the CA1 region may contain a portion of CA2. These ROIs are shown in Fig 6A and listed in the legend. Once the ROIs were drawn, we established an optical density threshold for each signal (CY3 and FITC) using the hippocampus MosaiX image from the IC on the same slide. We then measured the area occupied by each signal in each ROI and performed the same procedure on each image of the slide, keeping the same threshold parameters and following the same procedure. The data we obtained were the areas occupied by each signal expressed in pixels. It is important to clarify that Map2 signal was used as a guide to draw an ROI of the area surrounding the Map2 staining edges, defined with a threshold, and about a 10% enlargement to cover the possible area were the synaptophysin staining should be. For synaptophysin, the area measured was the actual staining area detected; the thresholds for both Map2 and Synaptophysin were systematically set in a cage control tissue from each slide and kept constant for the rest of the slide analysis. Then, the measures used for analysis was the area of synaptophysin staining divided by the ROI area defined by the Map2 staining x 100. And note that no differences were found in any ROI in the MAP2 defined area between groups (Fig 6B).

pone.0132676.g006: Synaptophysin/Map2 segmentation and Map2 staining area analysis.(A) A representative synaptophysin (Red) / Map2 (Green) stained image is shown with the drawings that defined the different hippocampal dendritic segments regions of interest (hippocampal segments ROIs). These hippocampal segments ROIs included the CA3 stratum oriens, divided into 3 regions based on their proximity to the dentate gyrus (DG): stratum oriens distal (SOd), stratum oriens medial (SOm), and stratum oriens proximal (SOp); the CA3 stratum lucidum was also divided into 3 regions, the stratum lucidum distal (SLd), stratum lucidum medial (SLm), and stratum lucidum proximal (SLp). The CA3 stratum radiatum was divided into 6 regions, depending on their proximity to the DG and to the pyramidal cell soma: stratum radiatum distal medial (SRdm), stratum radiatum medial medial (SRmm), stratum radiatum proximal medial (SRpm), stratum radiatum distal distal (SRdd), stratum radiatum medial distal (SRmd), and stratum radiatum proximal distal (SRpd). Finally, the last 2 ROIs correspond to the CA1 stratum oriens (CA1 SO) and the CA1 stratum radiatum (CA1 SR). In the bar graph (B) the Map2-stained area expressed in pixels is shown for each hippocampal segment ROI. It is important to emphasise that No significant differences were found among groups (IC, SC, and WM) in the Map2-stained area used for the synaptophysin analysis.

Mentions:
Analysis of the immunofluorescence for synaptophysin/Map2 staining was performed using ImageJ software (Freeware NIH USA). All images were pre-processed with the median filter for noise reduction. Then, each image was segmented into 14 different regions of interest (ROIs). The ROIs were drawn to segment different portions of the CA3 and CA1 dendritic regions (Fig 6A). In CA3, we distinguished between the stratum oriens and stratum lucidum, and between proximal and distal portions of the stratum radiatum based on their proximity to the CA3 pyramidal cell soma. The CA3 region was also divided into 3 regions (distal, medial, and proximal) based on their proximity to the DG granular layer (12 ROIs total in CA3). Finally, we divided the CA1 region between the stratum oriens (CA1 SO) and stratum radiatum (CA1 SR). It is possible that the 2 ROIs selected in the CA1 region may contain a portion of CA2. These ROIs are shown in Fig 6A and listed in the legend. Once the ROIs were drawn, we established an optical density threshold for each signal (CY3 and FITC) using the hippocampus MosaiX image from the IC on the same slide. We then measured the area occupied by each signal in each ROI and performed the same procedure on each image of the slide, keeping the same threshold parameters and following the same procedure. The data we obtained were the areas occupied by each signal expressed in pixels. It is important to clarify that Map2 signal was used as a guide to draw an ROI of the area surrounding the Map2 staining edges, defined with a threshold, and about a 10% enlargement to cover the possible area were the synaptophysin staining should be. For synaptophysin, the area measured was the actual staining area detected; the thresholds for both Map2 and Synaptophysin were systematically set in a cage control tissue from each slide and kept constant for the rest of the slide analysis. Then, the measures used for analysis was the area of synaptophysin staining divided by the ROI area defined by the Map2 staining x 100. And note that no differences were found in any ROI in the MAP2 defined area between groups (Fig 6B).

Bottom Line:
The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training.By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion.Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.

ABSTRACTSpatial water maze (WM) overtraining induces hippocampal mossy fiber (MF) expansion, and it has been suggested that spatial pattern separation depends on the MF pathway. We hypothesized that WM experience inducing MF expansion in rats would improve spatial pattern separation in the hippocampal network. We first tested this by using the the delayed non-matching to place task (DNMP), in animals that had been previously trained on the water maze (WM) and found that these animals, as well as animals treated as swim controls (SC), performed better than home cage control animals the DNMP task. The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training. Moreover, these behavioral treatments also enhance network reliability and improve partial pattern separation in CA1 and pattern completion in CA3. By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion. Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.